ApoA-I protects pancreatic β-cells from cholesterol-induced mitochondrial damage and restores their ability to secrete insulin

BACKGROUND: High cholesterol levels in pancreatic β-cells cause oxidative stress and decrease insulin secretion. β-cells can internalize apo (apolipoprotein) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves β-cell insulin secretion by reducing oxidative stress.

METHODS: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-β-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mmol/L glucose was quantified by radioimmunoassay. Internalization of fluorescently labeled apoA-I by β-cells was monitored by flow cytometry. The effects of apoA-I internalization on β-cell gene expression were evaluated by RNA sequencing. ApoA-I-binding partners on the β-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in β-cells and isolated islets with MitoSOX and confocal microscopy.

RESULTS: An F 1 -ATPase β-subunit on the β-cell surface was identified as the main apoA-I-binding partner. β-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol-, and F 1 -ATPase β-subunit-dependent. β-cells with internalized apoA-I (apoA-I + cells) had higher cholesterol and cell surface F 1 -ATPase β-subunit levels than β-cells without internalized apoA-I (apoA-I − cells). The internalized apoA-I colocalized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The IF 1 (ATPase inhibitory factor 1) attenuated apoA-I internalization and increased oxidative stress in Ins-1E β-cells and isolated mouse islets. Differentially expressed genes in apoA-I + and apoA-I − Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion, and mitochondrial function.

CONCLUSIONS: These results establish that β-cells are functionally heterogeneous, and apoA-I restores insulin secretion in β-cells with elevated cholesterol levels by improving mitochondrial redox balance.

Methods
Identification of the F1-ATPase β-subunit on the Ins-1E cell surface

Tosylactivated Dynabeads (Thermo Fisher Scientific) were used to identify apoA-I binding partners on the Ins-1E cell surface. Briefly, apoA-I (100 µg) was dissolved in borate buffer (0.1 M, pH 9.5) containing ammonium sulphate (1.2 M) and incubated at 37 °C overnight with Dynabeads (5 mg, 165 µL). The beads with bound apoA-I were collected using a magnet and coupling efficiency was determined by measuring the concentration of unbound apoA-I in the supernatant. The beads with bound apoA-I were resuspended in PBS (pH 7.4, 250 µL) with 0.1% (w/v) BSA and incubated for 5 min at room temperature on a rotary shaker. The beads were collected and resuspended in PBS (250 µL) with 0.5 % (w/v) BSA, then incubated for a further 1 h at 37 °C to reduce non-specific binding. The beads were collected again and resuspended in PBS (pH 7.4, 250 µL) without BSA.

Ins-1E cells were seeded in a 12-well plate, washed with KRBH buffer, then maintained at 4 °C for 1 h with KRBH with 0.1% (w/v) BSA (200 µL) and Dynabeads (50 µL) with bound apoA-I. The supernatant was discarded, and the cells were washed gently with ice-cold PBS, then harvested with PBS containing EDTA-Na2 (2 mM) and collected into an Eppendorf tube. The Dynabeads and the bound cells were collected with a magnet. The supernatant was removed and the beads were washed with PBS (pH 7.4) and 0.1% (v/v) Tween20, then washed with PBS (pH 7.4). Non-specifically bound proteins were removed from the Dynabeads by incubation overnight at room temperature with trypsin at protein to enzyme ratio of 100:1. The beads were then collected using a magnet, the supernatant was discarded and the beads were washed (x3) with PBS. ApoA-I binding partners on the beads were eluted with 0.15% (v/v) trifluoro acetic acid, concentrated using C18 stage tips as previously described and analysed by LC–MS/MS.

Mass Spectrometry

The eluted peptides were reconstituted in 0.1% (v/v) formic acid (10 μL) and resolved by nano-LC using an Ultimate 3000 HPLC and an autosampler (Dionex, Amsterdam, The Netherlands). Briefly, the sample (0.5 µL), was loaded onto a micro C18 pre-column (300 μm×5 mm, Dionex) with H2O:CH3CN (98:2) in 0.1% (v/v) trifluoro acetic acid at a flow rate of 10 μL/min. After washing, the pre-column was switched (Valco 10 port valve, Dionex) into line with a fritless nanocolumn (75 μm×22 cm) containing reverse phase C18 media (particle size: 1.9 μm, pore size: 120 Å, Dr. Maisch HPLC GmbH). Peptides were eluted using a linear gradient of H2O:CH3CN (98:2 to 64:36) in 0.1% (v/v) formic acid at a flow rate of 250 nL/min over 90 min. The QExactive (Thermo Electron, Bremen, Germany) mass spectrometer was run in DDA mode where 2000 V was applied to a low-volume union and the column (45 °C) was positioned 0.5 cm from the heated capillary (275 °C). A survey scan 350–1750 m/z was acquired in the Orbitrap (resolution 70,000 at 200 m/z) with an accumulation target of 106 ions, lock mass enabled and up to the 10 most abundant ions (AGC target set to 105, minimum AGC target set to 1.5×104) with charge states ≥ +2 and ≤ +6 sequentially isolated and fragmented.

Protein dataset-peak lists were generated from raw files using Mascot Daemon v2.5.1 (Matrix Science, London, UK, www.matrixscience.com). All MS/MS spectra were searched against the Uniprot database (Feb 2021) of 563,972 sequences for protein identification with the following criteria: (1) taxon, Rat; (2) allowed 1 missed cleavages; (3) variable modifications, oxidation (M), phosphorylation (S,T,Y), Carbomidomethyl (C); (4) peptide tolerance, ±5 ppm; (5) fragment tolerance, ±0.5 Da; (6) peptide charge +2 and +3; and (7) enzyme specificity, semi-tryptic. A decoy database search was also performed. Only proteins that had a significance threshold of p<0.007 (<1% FDR) were recorded.